Explore the vast range of titles available.

Abstract As research on plant viruses has focused mainly on crop diseases, little is known about these viruses in natural environments. To understand the ecology of viruses in natural systems, comprehensive information on virus–virus and virus–host interactions is required. We applied RNA-Seq to plants from a natural population of Arabidopsis halleri subsp. gemmifera to simultaneously determine the presence/absence of all sequence-reported viruses, identify novel viruses and quantify the host transcriptome. By introducing the criteria of read number and genome coverage, we detected infections by Turnip mosaic virus (TuMV), Cucumber mosaic virus and Brassica yellows virus. Active TuMV replication was observed by ultramicroscopy. De novo assembly further identified a novel partitivirus, Arabidopsis halleri partitivirus 1. Interestingly, virus reads reached a maximum level that was equivalent to that of the host's total mRNA, although asymptomatic infection was common. AhgAGO2, a key gene in host defence systems, was upregulated in TuMV-infected plants. Multiple infection was frequent in TuMV-infected leaves, suggesting that TuMV facilitates multiple infection, probably by suppressing host RNA silencing. Revealing hidden plant–virus interactions in nature can enhance our understanding of biological interactions and may have agricultural applications. RNA-Seq, coupled with de novo assembly, enables the comprehensive and solid determination of virus infection, and revealed the virus–virus and virus–host interactions in naturally growing plants.

P0 protein of some polerovirus members can target ARGONAUTE1 (AGO1) to suppress RNA silencing. Although P0 harbors an F-box-like motif reported to be essential for interaction with S phase kinase-associated protein 1 (SKP1) and RNA silencing suppression, it is the autophagy pathway that was shown to contribute to AGO1 degradation. Therefore, the role of P0–SKP1 interaction in silencing suppression remains unclear. We conducted global mutagenesis and comparative functional analysis of P0 encoded by Brassica yellows virus (BrYV) (P0Br). We found that several residues within P0Br are required for local and systemic silencing suppression activities. Remarkably, the F-box-like motif mutant of P0Br, which failed to interact with SKP1, is destabilized in vivo. Both the 26S proteasome system and autophagy pathway play a role in destabilization of the mutant protein. Furthermore, silencing of a Nicotiana benthamiana SKP1 ortholog leads to the destabilization of P0Br. Genetic analyses indicated that the P0Br–SKP1 interaction is not directly required for silencing suppression activity of P0Br, but it facilitates stability of P0Br to ensure efficient RNA silencing suppression. Consistent with these findings, efficient systemic infection of BrYV requires P0Br. Our results reveal a novel strategy used by BrYV for facilitating viral suppressors of RNA silencing stability against degradation by plant cells.

Studies on plant viruses are biased towards crop diseases and little is known about viruses in natural vegetation. We conducted extensive surveys of plant viruses in wild Brassicaceae plants occurring in three local plant communities in central Japan. We applied RNA-Seq with selective depletion of rRNA, which allowed us to detect infections of all genome-reported viruses simultaneously. Infections of Turnip mosaic virus (TuMV), Cucumber mosaic virus (CMV), Brassica yellows virus, Pelargonium zonate spot virus, and Arabidopsis halleri partitivirus 1 were detected from the two perennial species, Arabidopsis halleri subsp. gemmifera and Rorippa indica. De novo assembly further detected partial sequences of a putative novel virus in Arabis fragellosa. Virus species composition and infection rate differed depending on site and plant species. Viruses were most frequently detected from the perennial clonal plant, A. halleri, in which a high clonal transmission rate of viruses across multiple years was confirmed. Phylogenetic analysis of TuMVand CMV showed that virus strains from wild Brassicaceae were included as a major clade of these viruses with other reported strains from crop plants, suggesting that viruses were shared among wild plants and crops. Our studies indicated that distribution of viruses in natural plant populations are determined by the combinations of life histories of viruses and hosts. Revealing viral distribution in the natural plant communities improves our knowledge on the ecology of plant viruses.

P0 proteins encoded by poleroviruses Brassica yellows virus (BrYV) and Potato leafroll virus (PLRV) are viral suppressors of RNA silencing (VSR) involved in abolishing host RNA silencing to assist viral infection. However, other roles that P0 proteins play in virus infection remain unclear. Here, we found that C-terminal truncation of P0 resulted in compromised systemic infection of BrYV and PLRV. C-terminal truncation affected systemic but not local VSR activities of P0 proteins, but neither transient nor ectopic stably expressed VSR proteins could rescue the systemic infection of BrYV and PLRV mutants. Moreover, BrYV mutant failed to establish systemic infection in DCL2/4 RNAi or RDR6 RNAi plants, indicating that systemic infection might be independent of the VSR activity of P0. Partially rescued infection of BrYV mutant by the co-infected PLRV implied the functional conservation of P0 proteins within genus. However, although C-terminal truncation mutant of BrYV P0 showed weaker interaction with its movement protein (MP) when compared to wild-type P0, wild-type and mutant PLRV P0 showed similar interaction with its MP. In sum, our findings revealed the role of P0 in virus systemic infection and the requirement of P0 carboxyl terminal region for the infection.

The Brassica yellows virus genotype A P0 protein interacts with Rubisco assembly factor 2 in tobacco, affecting its nuclear accumulation and promoting viral infection. Abstract In interactions between poleroviruses and their hosts, few cellular proteins have been identified that directly interact with the multifunctional virus P0 protein. To help explore the functions of P0, we identified a Brassica yellows virus genotype A (BrYV-A) P0BrA-interacting protein from Nicotiana benthamiana, Rubisco assembly factor 2 (NbRAF2), which localizes in the nucleus, cell periphery, chloroplasts, and stromules. We found that its C-terminal domain (amino acids 183-211) is required for self-interaction. A split ubiquitin membrane-bound yeast two-hybrid system and co-immunoprecipitation assays showed that NbRAF2 interacted with P0BrA, and co-localized in the nucleus and at the cell periphery. Interestingly, the nuclear pool of NbRAF2 decreased in the presence of P0BrA and during BrYV-A infection, and the P0BrA-mediated reduction of nuclear NbRAF2 required dual localization of NbRAF2 in the chloroplasts and nucleus. Tobacco rattle virus-based virus-induced gene silencing of NbRAF2 promoted BrYV-A infection in N. benthamiana, and the overexpression of nuclear NbRAF2 inhibited BrYV-A accumulation. Potato leafroll virus P0PL also interacted with NbRAF2 and decreased its nuclear accumulation, indicating that NbRAF2 may be a common target of poleroviruses. These results suggest that nuclear NbRAF2 possesses antiviral activity against BrYV-A infection, and that BrYV-A P0BrA interacts with NbRAF2 and alters its localization pattern to facilitate virus infection.

RNA from a Chinese cabbage plant ( Brassica campestris ssp. pekinensis ) showing leaf malformation and mottling was labeled and hybridized to a DNA chip capable of detecting plant viruses and viroids. Probes specific for beet mild yellowing virus (BMYV) and beet western yellows virus (BWYV) yielded positive results, suggesting that the plant was infected by a polerovirus. Primers designed from the sequences of the positive probes were used to amplify and sequence one portion of the viral genome. This sequence showed a 90 % or greater identity to several poleroviruses, including BMYV, BWYV, beet chlorosis virus (BChV) and turnip yellows virus (TuYV). The complete genome sequence of the Chinese cabbage-infecting polerovirus consisted of 5,666 nt and was most closely related to brassica yellows virus (BrYV; 94 % identity). The virus was named BrYV-Cheongsong (BrYV-CS). However, ORF3, ORF4 and the 5′ half of ORF5 of BrYV-CS were more closely related to those of TuYV, BWYV, BChV and BMYV than to those of BrYV. Interestingly, a recombination event (positions 3531-4819 in BrYV-CS) was detected when this sequence was aligned with those of BrYV and TuYV. This region showed the highest sequence identity to that of TuYV (94 % identity) and had greater than 93 % identity to those of BWYV, BChV and BMYV, but it shared only 81 % identity with that of BrYV. Taken together, the genomes of BrYV-CS and BrYV are closely related. However, the structural genes in the 3′ half of the genome of BrYV-CS are more closely related to those of other poleroviruses.

Three novel viruses were detected from Japanese persimmon ‘Reigyoku’ (Diospyros kaki), which grows poorly when top-grafted onto some interstocks. The genomic characterization and the phylogenetic analysis of these viruses indicated that they are likely new members of the genera Ampelovirus, Polerovirus, and Waikavirus, tentatively named Persimmon ampelovirus (PAmpV), Persimmon polerovirus (PPolV), and Persimmon waikavirus (PWaiV). The alignment of two PAmpV variants with Plum bark necrosis stem pitting–associated virus isolates had 72.5%–79.0% amino acid sequence identities with RNA-dependent RNA polymerase, 71.6%–79.2% with a heat shock protein 70 homolog, and 66.2%–69.8% with coat protein, respectively. Two PPolV variants showed the highest amino acid sequence identities (46.6%) with the P1–P2 fusion protein of Brassica yellows virus. PWaiV had the highest amino acid sequence identity (41.9%) with the conserved domain between proteinase and polymerase of Rice tungro spherical virus. Reverse-transcription and polymerase chain reaction detected Persimmon virus A, Apple crinkle fruit viroid, and Citrus viroid VI from the mother clone with good growth, and PAmpV, PPolV, and Persimmon latent viroid (PLVd) additionally from 20 trees with poor growth. PAmpV, PPolV, and PLVd were detected from four, 10, and 10 of 14 trees with good growth, respectively, and the grafting of budwood harboring these three pathogens caused significant stunt in mother clones.

In 2018 virus-like symptoms, typical of polerovirus infection were observed in several oilseed rape crops in northern Greece. In order to identify the etiological agent of these symptoms a polerovirus-generic RT-PCR assay was applied. Sequencing of the amplicons revealed the presence of virus isolates genetically close to turnip yellows virus (TuYV). Further molecular characterization of the near complete genome of ‘1–2’, ‘Geo1’, ‘Geo7’ and ‘Geo15’ isolates revealed that they share > 96% nt identity with various TuYV sequences. On the other hand, the fifth, characterized isolate from oilseed rape, termed ‘1–1’, showed higher sequence similarity to brassica yellows virus (BrYV) regarding the 5′ part of the complete coding sequence, whereas the 3′ part was closely related to TuYV isolates. A recombination analysis using RDP indicated the presence of a putative breakpoint (nucleotide position 2964) in ‘1–1’ genome and it is proposed that the virus isolate ‘1–1’ might be an interspecies recombinant between BrYV and TuYV. To our knowledge, this is the first time that the complete coding sequences of Greek TuYV isolates have been determined and the first detection of a BrYV/TuYV recombinant isolate infecting oilseed rape in Greece.

microRNAs (miRNAs) influence many biological processes at the post-transcriptional level. However, the molecular characterization of miRNAs in the Myzus persicae response to Brassica yellows virus (BrYV) stress remains unclear. In this study, we present the results of miRNA profiling in Myzus persicae under two different treatments: treatment one (raised on turnip plants), and treatment two (raised on Arabidopsis thaliana). A total of 72 known and 113 novel mature miRNAs were identified in both non-viruliferous and viruliferous aphids, under treatment one. In treatment two, 72 known and 112 novel mature miRNAs were identified in BrYV-free aphids; meanwhile, 71 known and 115 novel miRNAs were identified in BrYV-carrying aphids. Moreover, eight upregulated and four downregulated miRNAs were identified in viruliferous aphids under treatment two, whereas only two miRNAs were differentially expressed under treatment one. These results indicated the relative BrYV level could influence miRNA expression in aphids. KEGG enrichment analysis showed the predicted genes targeted by differentially expressed miRNAs were primarily involved in Peroxisome, neuroactive ligand–receptor interaction, and metabolism of xenobiotics by cytochrome P450 pathways. Taken together, these findings reveal the effect of BrYV on miRNAs in Myzus persicae and provide key clues for further studies on the molecular mechanisms of BrYV transmission via aphids.

Viruses in the genus Polerovirus infect a wide range of crop plants and cause severe economic crop losses. BrYV belongs to the genus Polerovirus and is transmitted by Myzus persicae. However, the changes in transcriptome and proteome profiles of M. persicae during viral infection are unclear. Here, RNA-Seq and TMT-based quantitative proteomic analysis were performed to compare the differences between viruliferous and nonviruliferous aphids. In total, 1266 DEGs were identified at the level of transcription with 980 DEGs being upregulated and 286 downregulated in viruliferous aphids. At the protein level, among the 18 DEPs identified, the number of upregulated proteins in viruliferous aphids was twice that of the downregulated DEPs. Enrichment analysis indicated that these DEGs and DEPs were mainly involved in epidermal protein synthesis, phosphorylation, and various metabolic processes. Interestingly, the expressions of a number of cuticle proteins and tubulins were upregulated in viruliferous aphids. Taken together, our study revealed the complex regulatory network between BrYV and its vector M. persicae from the perspective of omics. These findings should be of great benefit to screening key factors involved in the process of virus circulation in aphids and provide new insights for BrYV prevention via vector control in the field.